The use of hematopoietic stem cells (HSC) in transplant biology and the potential use of these cells, which in the future may be derived by reprogramming methods, in regenerative medicine can be enhanced significantly by improving their engraftment and collection by mobilization. Maintenance of these functions critically depends on the interaction of HSC with one or several specialized microenvironments (so-called niches) in the bone marrow (BM). The crucial intracellular pathways triggered by these interactions are less well characterized, and how they are coordinated to regulate HSC localization relative to different components of the niche and subsequent survival and proliferation is not known. However, dissecting this complexity and defining the functional interactions between GTPases, the guanine exchange factors (GEFs) that activate them and effectors that subserve specific functions in HSC remains a major challenge because GEFs are promiscuous in vitro and effectors are cell and agonist-specific. We have identified the Rac GTPase signaling pathway as a critical integrating component of HSC engraftment and retention. This pathway determines both interaction with the HSC niche via regulation of the cytoskeleton and modulation of proliferation and survival of these cells via kinase pathway activation, potentially providing novel insights into the integration of these broad HSC functions. The long term goal of this work is to further identify a biochemical footprint of HSC marrow homing, retention and engraftment by taking advantage of the genetic models we have developed and validated in the previous funding period. Our central hypothesis is that Rac-dependent HSC regulation of homing/retention in the marrow and reconstitution of hematopoiesis is accomplished by specific functional modules of Vav and Pak proteins that distinctly regulate kinase activation, cell shape changes, and gene expression. This work is based on our latest understanding the role of the Rac signaling axis and development of reagents including mice that has been supported by this grant in the past decade. Our studies will define integral components of the Rac signaling axis that regulate HSC function, providing new knowledge of HSC homing, retention and engraftment as well as identifying new targets for potential therapeutic intervention in these processes. Since modulation of this pathway could improve engraftment of HSCs, lead to more effective mobilization of these cells and to the development of novel therapies in a variety of leukemias these studies are both innovative and translational in nature.